Multi-layer hydrogel capsules for encapsulation of cells and cell aggregates

ABSTRACT

Biomedical devices for implantation with decreased pericapsular fibrotic overgrowth are disclosed. The device includes biocompatible materials and has specific characteristics that allow the device to elicit less of a fibrotic reaction after implantation than the same device lacking one or more of these characteristic that are present on the device. Biocompatible hydrogel capsules encapsulating mammalian cells having a diameter of greater than 1 mm, and optionally a cell free core, are disclosed which have reduced fibrotic overgrowth after implantation in a subject. Methods of treating a disease in a subject are also disclosed that involve administering a therapeutically effective amount of the disclosed encapsulated cells to the subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Application No. 16/200,334,filed Nov. 26, 2018, which is a divisional of U.S. Application No.14/776,639, filed Sep. 14, 2015, now U.S. Pat. No. 10,172,791, issuedJan. 8, 2019, which is a National Phase application under U.S.C. § 371of International Application No. PCT/US2014/029189, filed Mar. 14, 2014,which is a continuation-in-part of U.S. Application No. 13/831,250,filed Mar. 14, 2013, now U.S. Patent No. 9,555,007, issued Jan. 31,2017, which are hereby incorporated herein by reference in theirentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under R01 DE016516awarded by National Institutes of Health. The Government has certainrights in the invention.

FIELD OF THE INVENTION

The invention is generally related to the field of fabrication and useof biomedical devices, including structural, therapeutic-containing, andcell-encapsulating devices. More specifically, some aspects of theinvention relate to improved physical parameters to ensure improvedbiocompatibility of implanted biomedical devices, includingbiocompatible hydrogel encapsulating mammalian cells and polymericparticles loaded with anti-inflammatory drugs.

BACKGROUND OF THE INVENTION

Biomaterials and devices transplanted in the body are being used for abroad spectrum of clinical applications such as cell transplantation,controlled drug release, continuous sensing and monitoring ofphysiological conditions, electronic pacing, and tissue regeneration.For these applications, the longevity and fidelity of the device ishighly dependent on its ability to ward off recognition by the hostimmune system. Immune recognition initiates a cascade of hostorchestrated cellular processes leading to foreign body reactions, whichinclude persistent inflammation, fibrosis (walling-off), and damage tothe surrounding tissue. These unwanted effects are both deleterious tothe function of the device and a significant cause of pain anddiscomfort for the patient.

In 1980, Lim and Sun, Science 210:908 (1980) introduced an alginatemicrocapsule coated with an alginate/polylysine complex forencapsulation of pancreatic islets. Hydrogel microcapsules have sincebeen extensively investigated for encapsulation of living cells or cellaggregates for tissue engineering and regenerative medicine (Orive etal., Nat. Medicine 9:104 (2003); Paul, et al., Regen. Med. 4:733 (2009);Read et al., Biotechnol. 19:29 (2001)). In general, capsules aredesigned to allow facile diffusion of oxygen and nutrients to theencapsulated cells, while releasing the therapeutic proteins secreted bythe cells, and to protect the cells from attack by the immune system.These have been developed as potential therapeutics for a range ofdiseases including type I diabetes, cancer, and neurodegenerativedisorders such as Parkinson's (Wilson et al., Adv. Drug. Deliv. Rev.60:124 (2008); Joki et al., Nat. Biotech. 19:35 (2001); Kishima et al.,Neurobiol. Dis. 16:428 (2004)). One of the most common capsuleformulations is based on alginate hydrogels, which can be formed throughionic crosslinking. In a typical process, the cells are first blendedwith a viscous alginate solution. The cell suspension is then processedinto micro-droplets using different methods such as air shear, acousticvibration or electrostatic droplet formation (Rabanel et al.,Biotechnol. Prog. 25:946 (2009)). The alginate droplet is gelled uponcontact with a solution of divalent ions, such as Ca2+ or Ba2+.

One challenge associated with alginate microcapsules for cellencapsulation, however, is the lack of control of the relative positionsof the cells within the capsules. The cells can become trapped andexposed on the capsule surface, leading to inadequate immune-protection(Wong et al., Biomat. Artific. Cells Immobiol. Biotechnol. 19:675(1991)). It has been recognized that incomplete coverage would not onlycause the rejection of exposed cells but may also allow the infiltrationof macrophages and fibroblasts into the capsules through the exposedareas (Chang, Nat, Rev. Drug Discovery 4:221 (2005)). Alginate hydrogelmicrocapsules have been broadly investigated for their utility withpancreatic islets to treat Type I diabetes (Calafiore, Expert Opin.Biol. Ther. 3:201 (2003)). Numerous promising results have been reportedin several animal models including rodents (Lim, Science 210:908 (1980);Qi et al., Artifi. Cells, Blood Substitutes, Biotechnol. 36:403 (2008)),dogs (Soon-Shiong et al., Proc. Natl. Acad. Sci. USA 90:5843 (1993)),and nonhuman primates (El, X. Ma, D. Zhou, I. Vacek, A. M. Sun. J. Clin.Invest. 98:417 (1996)). Clinical trials have also been performed bySoon-Shiong et al., Lancet 343:950 (1994); Elliott et al.,Xenotransplantation 14:157 (2007); Calafiore et al., Diabetes Care29:137 (2006), Basta et al., Diabetes Care 34:2406 (2011); and Tuch etal., Diabetes Care 32:1887 (2009). In general, these clinical trialshave reported insulin secretion, but without long term correction ofblood sugar control, and additional challenges remain to advance thesesystems (Tam et al., J. Biomed. Mater. Res. Part A 98A:40 (2011); deVoset al., Biomaterials 27:5603 (2006)).

One challenge is the biocompatibility of the capsules. Upontransplantation, the foreign body responses cause fibrotic cellularovergrowth on the capsules that cut off the diffusion of oxygen andnutrients, and lead to necrosis of encapsulated islets. To this end,research groups have developed polymers to reduce the fibroticreactions. (Ma et al., Adv. Mater. 23:H189 (2011)). Another challenge isthe incomplete coverage of the islets within the capsules (deVos et al.,Transplantation 62:888 (1996); deVos et al., Transplantation 62:893(1996)). Islets protruding outside the capsules are more frequentlyobserved when the islet number density in alginate solution increases orthe capsule size decreases, both of which are desirable to minimize thetransplantation volume (Leung, et al, Biochem. Eng. J. 48:337 (2010)).It has been hypothesized that if even a small fragment of islet isexposed, immune effector cells may destroy the entire islet (King etal., Graft 4:491 (2001); Weber et al., Ann. NY Acad. Sci. 875:233(1999)). Furthermore, exposure of a small number of islets may start acascade of events that leads to enhanced antigen-specific cellularimmunity and transplant failure.

A double-encapsulation process (Elliot, 2007; Wong et al., Biomat.Artific. Cells Immobiol. Biotechnol. 19:687 (1991)) has been used toimprove the encapsulation and xenograft survival where very smallcapsules containing cells were first formed and then several smallcapsules were enclosed in each larger capsule. This approach requiredtwo process steps and the large size of the final capsules inevitablylimited the mass transport that was essential to cell viability andfunctionality. As reported by Schneider et al. (Biomaterials22(14):1961-1970 (2001)), islet containing alginate beads were coatedwith alternating layers of polyethyleneimine, polyacrylacid orcarboxymethylcellulose and alginate to address some of these problems.Thin conformal coating of islets reduces the diffusion distance andtotal transplantation volume (Teramura et al., Adv. Drug Deliv. Rev.62:827 (2010); Wilson et al., J. Am. Chem. Soc. 131:18228 (2009)).However, the process often involves multiple steps which cause damage toislets and it is not clear whether the coatings are sufficiently robustfor clinical use (Califiore, 2003; Basta et al., Curr. Diab. Rep. 11:384(2011)). Previous data by Basta et al. (Transpl. Immunol. 13:289 (2004))has suggested conformal coatings may have reduced immune-protectivecapacity compared with the hydrogel capsules.

In summary, despite promising studies in various animal models over manyyears, encapsulated human islets so far have not made an impact in theclinical setting. Many non-immunological and immunological factors suchas biocompatibility, reduced immunoprotection, hypoxia, pericapsularfibrotic overgrowth, effects of the encapsulation process, andpost-transplant inflammation hamper the successful application of thispromising technology (Vaithilinga et al., Diabet Stud 8(1):51-67(2011)). Currently used alginate microcapsules often have isletsprotruding outside capsules, leading to inadequate immunoprotection.Improved encapsulation using a two-fluid co-axial electro jetting methodto confine islets in the core region of the capsules has been reportedby Ma et al., Adv. Healthcare Materials 2(5):667-672 (2013).

One major challenge to clinical application of encapsulated cells andother biomaterials and medical devices is their potential to induce anon-specific host response (Williams, Biomaterials 29(20):2941-53(2008); Park et al., Pharm Res 13(12):1770-6 (1996); Kvist et al.,Diabetes Technol 8(4):463-75 (2006); Wisniewski et al., J Anal Chem366(6):611-21 (2000); Van der Giessen et al., Circulation 94(7):1690-7(1996); Granchi et al., J Biomed Mater Res 29(2):197-202 (1995); Ward etal., Obstet Gynecol 86(5):848-50 (1995); Remes et al., Biomaterials13(11):731-43 (1992)). This reaction involves the recruitment of earlyinnate immune cells such as neutrophils and macrophages, followed byfibroblasts which deposit collagen to form a fibrous capsule surroundingthe implanted object (Williams, Biomaterials 29(20):2941-53 (2008);Remes et al., Biomaterials 13(11):731-43 (1992); Anderson et al., SeminImmunol 20(2):86-100 (2008); Anderson et al., Adv Drug Deliver Rev28(1):5-24 (1997); Abbas et al., Pathologic Basis of Disease. 7th ed.Philadelphia: W. B Saunders (2009)). Fibrotic cell layers can hinderelectrical (Singarayar et al., PACE 28(4):311-5 (2005)) or chemicalcommunications and prevent transport of analytes (Sharkawy et al., JBiomed Mater Res 37(3):401-12 (1997); Sharkawy et al., J Biomed MaterRes 40(4):598-605 (1998); Sharkawy et al., J Biomed Mater Res40(4):586-97 (1998)) and nutrients, thus leading to the eventual failureof many implantable medical devices such as immunoisolated pancreaticislets (De Groot et al., J Surg Res 121(1):141-50 (2004); De Vos et al.,Diabetologia 40(3):262-70 (1997); Van Schilfgaarde et al., J Mol Med77(1):199-205 (1999)).

The fibrotic reactions to the capsules upon transplantation pose a majorchallenge to transplanting islets. The fibrosis eventually leads tonecrosis of islets and failure of transplant.

There remains a substantial need for improved encapsulation methods anddevices for transplantation of human islet cells.

It is an object of the present invention to provide a cell encapsulationsystem for transplanting cells with reduced pericapsular fibroticovergrowth.

It is a further object of the invention to provide a method fortransplanting cells with reduced pericapsular fibrotic overgrowth.

It is a further object of the invention to provide improved methods fortreating diabetes using encapsulated islet cells.

It is a further object of the invention to provide a method and devicesfor general device fabrication to prevent fibrotic sequestration andprevention of function and long-term therapeutic efficacy.

SUMMARY OF THE INVENTION

It has been discovered that biomedical devices designed with certainphysical characteristics exhibit reduced host rejection and fibroticovergrowth of biomaterials and biomedical devices.

A biomedical device for implantation with decreased pericapsularfibrotic overgrowth has been developed. The device includesbiocompatible materials, has a diameter of at least 1 mm and less than10 mm, has a spheroid-like shape, and has one or more of the additionalcharacteristics: surface pores of the device are greater than 0 nm andless than 10 μm; the surface of the device is neutral or hydrophilic;the curvature of the surface of the device is at least 0.2 and is notgreater than 2 on all points of the surface; and the surface of thedevice does not have flat sides, sharp angles, grooves, or ridges. Thedevice elicits less of a fibrotic reaction after implantation than thesame device lacking one or more of these characteristic that are presenton the device. In some embodiments, the devices can be a biocompatiblesphere-like (spheroid) outer shell, encapsulating, for example, biologiccells or tissue, synthetic electrical leads, or sensors.

The biomedical device can be made from a variety of materials, includinghydrogels, both chemically purified as well as endotoxin-containingfood-grade alginate, semiconducting materials, such assilicon-dioxide-based ceramics like glass, and degradable andnon-degradable polymers and plastics, such as PCL and polystyrene. Thespecific combinations of features result in or improve biocompatibilityof devices (in particular, by reducing or eliminating immune reaction toand capsular fibrosis of the devices).

The biomedical device can be used for any biomedical application,including, for example, long-term implantable sensors and actuators,controlled drug releasing devices, prostheses and tissue regeneratingbiomaterials, and technologies pertaining to transplantation.

A biocompatible capsule encapsulating mammalian cells, optionallyincluding anti-inflammatory drugs, for transplantation with decreasedpericapsular fibrotic overgrowth has been developed. The capsule hasencapsulated therein mammalian cells. The mammalian cells are notlocated in the core of the capsule. Preferably the capsule contains twoor three layers. In tri-layer capsules, having an acellular core, ahydrogel layer, and an outer barrier layer, the cells are located in themiddle layer. For two layer capsules, which contain a core and a shell,the cells are located in the shell. Alternatively, the cells can be inthe core of a two layer capsule. Optionally, the capsule also containsone or more therapeutic, diagnostic and/or prophylactic agents, such asanti-inflammatory drugs or radiopaque imaging agents, encapsulatedtherein.

Generally, the core-shell capsules are fabricated without a membranelayer using a microfluidic or needle system to form capsules andmicrocapsules with two or more integrated layers. For example, twoconcurrent liquid streams may be used to form two-layer droplets. Thetri-layer capsules can be fabricated without a membrane layer. Forexample, three concurrent streams may be used to form three-layerdroplets that harden to form the capsules and microcapsules.

Cells suitable for encapsulation and transplantation are generallysecretory or metabolic cells (i.e., they secrete a therapeutic factor ormetabolize toxins, or both) or structural cells (e.g., skin, muscle,blood vessel), or metabolic cells (i.e., they metabolize toxicsubstances). In some embodiments, the cells are naturally secretory,such as islet cells that naturally secrete insulin, or naturallymetabolic, such as hepatocytes that naturally detoxify and secrete. Insome embodiments, the cells are bioengineered to express a recombinantprotein, such as a secreted protein or metabolic enzyme. Depending onthe cell type, the cells may be organized as single cells, cellaggregates, spheroids, or even natural or bioengineered tissue.

The capsule is preferably a hydrogel capsule. In some embodiments thecore of the capsule is a non-hydrogel polymer. Preferably the capsuleshave a diameter greater than 1 mm, more preferably the diameter is 1.5mm or greater in size, but less than 8 mm. The larger capsules requirean acellular core to ensure that the cells are able to receive adequatenutrients and gas exchange by diffusion throughout the hydrogel.

A population of hydrogel capsules encapsulating cells has beendeveloped. Capsules in the population contain cells to be transplantedselected from the group consisting of mammalian secretory cells,mammalian metabolic cells, mammalian structural cells, and aggregatesthereof. All of the capsules in the population of capsules have adiameter of at least 1 mm and less than 8 mm. The capsules in thepopulation elicit less of a fibrotic reaction after implantation thanthe same capsules having a diameter of less than 1 mm. The capsules inthe population can be selected by separating capsules having a diameterof at least 1 mm and less than 8 mm from capsules having a diameter ofless than 1 mm and from capsules having a diameter of at least 8 mm.

The compositions may be fabricated into artificial organs, such as anartificial pancreas containing encapsulated islet cells. In some ofthese embodiments, the cells are encapsulated in a single hydrogelcompartment. In other embodiments, the composition contains a pluralityof encapsulated cells dispersed or encapsulated in a biocompatiblestructure.

Methods for treating diseases generally involve administering to asubject a biocompatible hydrogel encapsulating mammalian cells andanti-inflammatory drugs. In some embodiments, the anti-inflammatorydrugs are encapsulated in controlled release polymer. In some of theseembodiments, the encapsulated cells preferably secrete a therapeuticallyeffective amount of a substance to treat the disease for at least 30days, preferably at least 60 days, more preferably at least 90 days. Inparticularly preferred embodiments, the cells are islet cells thatsecrete a therapeutically effective amount of insulin to treat diabetesin the subject for at least 30 days, preferably at least 60 days, morepreferably at least 90 days.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of a tri-layer capsule, with the cellsencapsulated in a core hydrogel and the drug-loaded particles containedin an outer (envelope) hydrogel. An optional membrane material is shownseparating the core and envelope hydrogels.

FIG. 2 is an illustration of a nozzle for forming tri-layer capsules viatri-fluid coaxial electrospraying.

FIGS. 3A and 3B are schematic depictions of a conventional cellencapsulation method in which cells are dispersed within a hydrogelmatrix having an outer crosslinked shell (FIG. 3A) and a two-fluidco-axial electro jetting device for forming a hydrogel core containingcells, surrounded by a shell to prevent any of the cells from contactingthe outer wall of the capsule and potentially being exposed to immunecells in the host into which the capsule is implanted.

FIGS. 4A and 4B are graphs comparing control, regular capsules andcore-shell capsules in treating Type I diabetes. FIG. 4A shows the bloodglucose data of STZ-induced diabetic mice after the transplantation of500 encapsulated rat islet equivalents. The error bars representstandard errors. FIG. 4B shows the number of normoglycemic mice as afunction of time. 8 replicates were used for both types of capsules.

FIGS. 5A and 5B are graphs comparing blood glucose levels in STZ-inducedC57BL/6 diabetic mice implanted with 500 μm hydrogel capsules containingislet cells (FIG. 5A) and 1.5 mm alginate capsules encapsulating ratislets (500 IE's) (FIG. 5B).

FIG. 6 is a graph of immune response (percent of cell population made upof macrophages or neutrophils) in empty capsules of different sizes.Capsules of 300 μm, 500 μm, 900 μm, and 1500 μm were assessed.

FIG. 7 is a graph of normalized signal intensity of smooth muscle actinand β-actin associated with capsules of 0.3 mm, 0.5 mm, 1 mm, and 1.5 mmdetermined by Western blot.

FIG. 8 shows graphs of the fibrosis level for capsules of 300 μm, 500μm, and 800 μm normalized for surface area of the capsules (FIG. 8, top)or for volume of the capsules (FIG. 8, bottom).

FIGS. 9A-9D are graphs of the relative expression of fibrosis markers incapsules of 500 μm and 1500 μm were made of SLG20 alginate (PRONOVA®),polystyrene, glass, polycaprolactone (PCL), and LF10/60 alginate (FMCBIOPOLYMER®). Fibrosis was measured using CD68 (macrophage marker; FIG.9A), Ly6g (neutrophil marker; FIG. 9B), CD11b (myeloid cell marker; FIG.9C), and Collal (fibrosis marker; FIG. 9D).

FIGS. 10A and 10B are graphs of immune response (percent of cellpopulation made up of macrophages (FIG. 10A) or neutrophils (FIG. 10B))in capsules of 500 μm and 1500 μm made of different materials. Thecapsules were made of SLG20 alginate (PRONOVA®), polystyrene, glass,polycaprolactone (PCL), and LF10/60 alginate (FMC BIOPOLYMER®).

FIGS. 10A and 10B are graphs of immune response (percent of cellpopulation made up of macrophages (FIG. 10A) or neutrophils (FIG. 10B))in capsules of 500 μm and 1500 μm made of different materials. Thecapsules were made of SLG20 alginate (PRONOVA), polystyrene, glass,polycaprolactone (PCL), and LF10/60 alginate (FMC BioPolymer).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

“Hydrogel” refers to a substance formed when an organic polymer (naturalor synthetic) is cross-linked via covalent, ionic, or hydrogen bonds tocreate a three-dimensional open-lattice structure which entraps watermolecules to form a gel. Biocompatible hydrogel refers to a polymer thatforms a gel which is not toxic to living cells, and allows sufficientdiffusion of oxygen and nutrients to the entrapped cells to maintainviability.

“Alginate” is a collective term used to refer to linear polysaccharidesformed from β-D-mannuronate and α-L-guluronate in any M/G ratio, as wellas salts and derivatives thereof. The term “alginate”, as used herein,encompasses any polymer having the structure shown below, as well assalts thereof.

“Biocompatible” generally refers to a material and any metabolites ordegradation products thereof that are generally non-toxic to therecipient and do not cause any significant adverse effects to thesubject.

“Biodegradable” generally refers to a material that will degrade orerode by hydrolysis or enzymatic action under physiologic conditions tosmaller units or chemical species that are capable of being metabolized,eliminated, or excreted by the subject. The degradation time is afunction of polymer composition and morphology.

“Drug-loaded particle” refers to a polymeric particle having a drugdissolved, dispersed, entrapped, encapsulated, or attached thereto.

“Microparticle” and “nanoparticle” refer to a polymeric particle ofmicroscopic and nanoscopic size, respectively, optionally containing adrug dissolved, dispersed, entrapped, encapsulated, or attached thereto.

“Anti-inflammatory drug” refers to a drug that directly or indirectlyreduces inflammation in a tissue. The term includes, but is not limitedto, drugs that are immunosuppressive. The term includesanti-proliferative immunosuppressive drugs, such as drugs that inhibitthe proliferation of lymphocytes.

“Immunosuppressive drug” refers to a drug that inhibits or prevents animmune response to a foreign material in a subject Immunosuppressivedrugs generally act by inhibiting T-cell activation, disruptingproliferation, or suppressing inflammation. A person who is undergoingimmunosuppression is said to be immunocompromised.

“Mammalian cell” refers to any cell derived from a mammalian subjectsuitable for transplantation into the same or a different subject. Thecell may be xenogeneic, autologous, or allogeneic. The cell can be aprimary cell obtained directly from a mammalian subject. The cell mayalso be a cell derived from the culture and expansion of a cell obtainedfrom a subject. For example, the cell may be a stem cell Immortalizedcells are also included within this definition. In some embodiments, thecell has been genetically engineered to express a recombinant proteinand/or nucleic acid.

“Autologous” refers to a transplanted biological substance taken fromthe same individual.

“Allogeneic” refers to a transplanted biological substance taken from adifferent individual of the same species.

“Xenogeneic” refers to a transplanted biological substance taken from adifferent species.

“Islet cell” refers to an endocrine cell derived from a mammalianpancreas. Islet cells include alpha cells that secrete glucagon, betacells that secrete insulin and amylin, delta cells that secretesomatostatin, PP cells that secrete pancreatic polypeptide, or epsiloncells that secrete ghrelin. The term includes homogenous andheterogenous populations of these cells. In preferred embodiments, apopulation of islet cells contains at least beta cells.

“Transplant” refers to the transfer of a cell, tissue, or organ to asubject from another source. The term is not limited to a particularmode of transfer. Encapsulated cells may be transplanted by any suitablemethod, such as by injection or surgical implantation.

“Sphere-like shape” refers to an object having a surface that roughlyforms a sphere. Beyond a perfect or classical sphere shape, asphere-like shape can have waves and undulations. Generally, asphere-like shape is an ellipsoid (for its averaged surface) withsemi-principal axes within 10% of each other. The diameter of asphere-like shape is the average diameter, such as the average of thesemi-principal axes.

“Spheroid-like shape” refers to an object having a surface that roughlyforms a spheroid. Beyond a perfect or classical sphere, oblate spheroid,or prolate spheroid shape, a spheroid-like shape can have waves andundulations. Generally, a spheroid-like shape is an ellipsoid (for itsaveraged surface) with semi-principal axes within 100% of each other.The diameter of a spheroid-like shape is the average diameter, such asthe average of the semi-principal axes.

“Flat side” refers to a contiguous area of more than 5% of a surfacethat has a curvature of 0.

“Sharp angle” refers to a location on a surface across which the tangentto the surface changes by more than 10% over a distance of 2% or less ofthe circumference of the surface. Edges, corners, grooves, and ridges ina surface are all forms of sharp angles.

II. Biomedical Devices and Capsules

A. Biomedical Devices with Reduced Fibrosis

Biomedical devices are disclosed for implantation into a subject. Thedevices are formed from biocompatible materials, such as biocompatiblepolymers, biodegradable and non-biodegradable polymers, semiconductormaterials, ceramics, and glass. In order to reduce or prevent fibrosis,the devices should include some or all of a suite of characteristics:

(1) Size: The device should have a minimum diameter of 1 mm (preferable1.5 mm). The diameter can be increased upwards of 8 to 10 mm.

(2) Shape: The overall device should be a sphere, sphere-like, spheroid,or spheroid-like. The curvature at any point on the surface of thedevice should be no less than 0.2 and no more than 2. Surface pores areignored for the purpose of calculating the curvature of the surface ofthe device.

(3) Hydrophobicity: The surface of the device should either be neutralor more water-loving (hydrophilic), and should not have a hydrophobicchemistry, such as with Teflon.

(4) Surface pore size: Pores on the surface of the device should beabove 0 nm but should not exceed 10 μm.

(5) Surface topography: The device should not have any flat sides, sharpangles, grooves, or ridges.

These characteristics have been discovered to affect the fibrosis ofimplanted devices. Devices having the first two characteristics and atleast one of the other characteristics have been shown to exhibitreduced fibrosis. Devices can be made with any one or combination ofthese characteristics. Devices having all of these characteristics arepreferred. Generally, the disclosed devices will elicit less of afibrotic reaction after implantation than the same device having any oneor more of these characteristics. In some embodiments, the device willelicit less of a fibrotic reaction after implantation than the samedevice having the first two characteristics and at least one of theother characteristics. In some embodiments, the device will elicit lessof a fibrotic reaction after implantation than the same device havingall of the characteristics. In some embodiments, the biomedical devicesare capsules or hydrogel capsules as described herein. Reference hereinto a capsule or a hydrogel capsule is intended to also refer to abiomedical device of the same description as the referenced capsule.

The disclosed devices have advantages and improvements over existingdevices and materials. For example, it has been discovered that objectswith flat surfaces (curvature of zero), such as cylinders, becomefibrosed even if they are large in overall size (over 1 mm). On theother extreme, objects with a high curvature (and thus having too smalla diameter, i.e., less than 1 mm), are capable of high packing densitieswith other transplanted materials and/or surrounding tissue, whichfacilitates immune cell adherence and fibrotic deposition. It has beenestablished that the specific combination of features together reduce orprevent fibrosis of many material classes, including hydrogels (bothchemically purified as well as endotoxin-containing food-gradealginate), semiconducting materials (such as silicon-dioxide-basedceramics like glass), and degradable and non-degradable polymers andplastics (such as PCL and polystyrene). Thus, the disclosed devices canbe made of these and a wide variety of other materials.

Hydrogel alginate spheres with the identified characteristics have beengenerated and demonstrated to prevent fibrosis and ensure long-termviability and functionality of transplanted mammalian cells of variousorigin (i.e., rat, mouse, pig, human) for treatment of disease (type 1diabetes). Optionally, the shell of the device may also contain one ormore therapeutic agents (such as anti-inflammatories) or diagnosticagents (such as oxygen sensing or imaging chemistries).

The biomedical device can be used for any biomedical application,including, for example, long-term implantable sensors and actuators,controlled drug releasing devices, prostheses and tissue regeneratingbiomaterials, and technologies pertaining to implantation andtransplantation. Many biomedical devices and purposes are known and thedisclosed devices can be adapted for such purposes.

B. Encapsulated Cells with Reduced Fibrosis

Capsules are disclosed for transplanting mammalian cells into a subject.The capsules are formed from a biocompatible, hydrogel-forming polymerencapsulating the cells to be transplanted. In order to inhibit capsularovergrowth (fibrosis), the structure of the capsules prevents cellularmaterial from being located on the surface of the capsule. Additionally,the structure of the capsules ensures that adequate gas exchange occurswith the cells and nutrients are received by the cells encapsulatedtherein. Optionally, the capsules also contain one or moreanti-inflammatory drugs encapsulated therein for controlled release.

C. Biocompatible Polymers

The disclosed compositions are formed from a biocompatible,hydrogel-forming polymer encapsulating the cells to be transplanted.Examples of materials which can be used to form a suitable hydrogelinclude polysaccharides such as alginate, collagen, chitosan, sodiumcellulose sulfate, gelatin and agarose, water soluble polyacrylates,polyphosphazines, poly(acrylic acids), poly(methacrylic acids),poly(alkylene oxides), poly(vinyl acetate), polyvinylpyrrolidone (PVP),and copolymers and blends of each. See, for example, U.S. Pat. Nos.5,709,854, 6,129,761 and 6,858,229.

In general, these polymers are at least partially soluble in aqueoussolutions, such as water, buffered salt solutions, or aqueous alcoholsolutions, that have charged side groups, or a monovalent ionic saltthereof Examples of polymers with acidic side groups that can be reactedwith cations are poly(phosphazenes), poly(acrylic acids),poly(methacrylic acids), poly(vinyl acetate), and sulfonated polymers,such as sulfonated polystyrene. Copolymers having acidic side groupsformed by reaction of acrylic or methacrylic acid and vinyl ethermonomers or polymers can also be used. Examples of acidic groups arecarboxylic acid groups and sulfonic acid groups.

Examples of polymers with basic side groups that can be reacted withanions are poly(vinyl amines), poly(vinyl pyridine), poly(vinylimidazole), and some imino substituted polyphosphazenes. The ammonium orquaternary salt of the polymers can also be formed from the backbonenitrogens or pendant imino groups. Examples of basic side groups areamino and imino groups.

The biocompatible, hydrogel-forming polymer is preferably awater-soluble gelling agent. In preferred embodiments, the water-solublegelling agent is a polysaccharide gum, more preferably a polyanionicpolymer.

The cells are preferably encapsulated using an anionic polymer such asalginate to provide the hydrogel layer (e.g., core), where the hydrogellayer is subsequently cross-linked with a polycationic polymer (e.g., anamino acid polymer such as polylysine) to form a shell. See e.g., U.S.Pat. Nos. 4,806,355, 4,689,293 and 4,673,566 to Goosen et al.; U.S. Pat.Nos. 4,409,331, 4,407,957, 4,391,909 and 4,352,883 to Lim et al.; U.S.Pat. Nos. 4,749,620 and 4,744,933 to Rha et al.; and U.S. Pat. No.5,427,935 to Wang et al., Amino acid polymers that may be used tocrosslink hydrogel forming polymers such as alginate include thecationic poly(amino acids) such as polylysine, polyarginine,polyornithine, and copolymers and blends thereof.

1. Polysaccharides

Several mammalian and non-mammalian polysaccharides have been exploredfor cell encapsulation. Exemplary polysaccharides suitable for cellencapsulation include alginate, chitosan, hyaluronan (HA), andchondroitin sulfate. Alginate and chitosan form crosslinked hydrogelsunder certain solution conditions, while HA and chondroitin sulfate arepreferably modified to contain crosslinkable groups to form a hydrogel.

In preferred embodiments, the biocompatible, hydrogel-forming polymerencapsulating the cells is an alginate. Alginates are a family ofunbranched anionic polysaccharides derived primarily from brown algaewhich occur extracellularly and intracellularly at approximately 20% to40% of the dry weight. The 1,4-linked α-1-guluronate (G) andβ-d-mannuronate (M) are arranged in homopolymeric (GGG blocks and MMMblocks) or heteropolymeric block structures (MGM blocks). Cell walls ofbrown algae also contain 5% to 20% of fucoidan, a branchedpolysaccharide sulphate ester with 1-fucose four-sulfate blocks as themajor component. Commercial alginates are often extracted from algaewashed ashore, and their properties depend on the harvesting andextraction processes.

Alginate forms a gel in the presence of divalent cations via ioniccrosslinking. Although the properties of the hydrogel can be controlledto some degree through changes in the alginate precursor (molecularweight, composition, and macromer concentration), alginate does notdegrade, but rather dissolves when the divalent cations are replaced bymonovalent ions. In addition, alginate does not promote cellinteractions.

A particularly preferred composition is a capsule or microcapsulecontaining cells immobilized in a core of alginate with a polylysineshell. Preferred capsules and microcapsules may also contain anadditional external alginate layer (e.g., envelope) to form amulti-layer alginate/polylysine-alginate/alginate-cells capsule ormicrocapsule. See U.S. Pat. No. 4,391,909 to Lim et al., for descriptionof alginate hydrogel crosslinked with polylysine. Other cationicpolymers suitable for use as a cross-linker in place of polylysineinclude poly(β-amino alcohols) (PBAAs) (Ma M et al., Adv. Mater.23:H189-94 (2011).

Chitosan is made by partially deacetylating chitin, a naturalnonmammalian polysaccharide, which exhibits a close resemblance tomammalian polysaccharides, making it attractive for cell encapsulation.Chitosan degrades predominantly by lysozyme through hydrolysis of theacetylated residues. Higher degrees of deacetylation lead to slowerdegradation times, but better cell adhesion due to increasedhydrophobicity. Under dilute acid conditions (pH<6), chitosan ispositively charged and water soluble, while at physiological pH,chitosan is neutral and hydrophobic, leading to the formation of a solidphysically crosslinked hydrogel. The addition of polyol salts enablesencapsulation of cells at neutral pH, where gelation becomes temperaturedependent.

Chitosan has many amine and hydroxyl groups that can be modified. Forexample, chitosan has been modified by grafting methacrylic acid tocreate a crosslinkable macromer while also grafting lactic acid toenhance its water solubility at physiological pH. This crosslinkedchitosan hydrogel degrades in the presence of lysozyme and chondrocytes.Photopolymerizable chitosan macromer can be synthesized by modifyingchitosan with photoreactive azidobenzoic acid groups. Upon exposure toUV in the absence of any initiator, reactive nitrene groups are formedthat react with each other or other amine groups on the chitosan to forman azo crosslink.

Hyaluronan (HA) is a glycosaminoglycan present in many tissuesthroughout the body that plays an important role in embryonicdevelopment, wound healing, and angiogenesis. In addition, HA interactswith cells through cell-surface receptors to influence intracellularsignaling pathways. Together, these qualities make HA attractive fortissue engineering scaffolds. HA can be modified with crosslinkablemoieties, such as methacrylates and thiols, for cell encapsulation.Crosslinked HA gels remain susceptible to degradation by hyaluronidase,which breaks HA into oligosaccharide fragments of varying molecularweights. Cells can be encapsulated in photopolymerized HA hydrogelswhere the gel structure is controlled by the macromer concentration andmacromer molecular weight. In addition, photopolymerized HA and dextranhydrogels maintain long-term culture of undifferentiated human embryonicstem cells. HA hydrogels have also been fabricated through Michael-typeaddition reaction mechanisms where either acrylated HA is reacted withPEG-tetrathiol, or thiol-modified HA is reacted with PEG diacrylate.

Chondroitin sulfate makes up a large percentage of structuralproteoglycans found in many tissues, including skin, cartilage, tendons,and heart valves, making it an attractive biopolymer for a range oftissue engineering applications. Photocrosslinked chondroitin sulfatehydrogels can be been prepared by modifying chondroitin sulfate withmethacrylate groups. The hydrogel properties were readily controlled bythe degree of methacrylate substitution and macromer concentration insolution prior to polymerization. Further, the negatively chargedpolymer creates increased swelling pressures allowing the gel to imbibemore water without sacrificing its mechanical properties. Copolymerhydrogels of chondroitin sulfate and an inert polymer, such as PEG orPVA, may also be used.

2. Synthetic Polymers

Polyethylene glycol (PEG) has been the most widely used syntheticpolymer to create macromers for cell encapsulation. A number of studieshave used poly(ethylene glycol) di(meth)acrylate to encapsulate avariety of cells. Biodegradable PEG hydrogels can be been prepared fromtriblock copolymers of poly(α-hydroxy esters)-b-poly (ethyleneglycol)-b-poly(α-hydroxy esters) endcapped with (meth)acrylatefunctional groups to enable crosslinking. PLA and poly(8-caprolactone)(PCL) have been the most commonly used poly(α-hydroxy esters) increating biodegradable PEG macromers for cell encapsulation. Thedegradation profile and rate are controlled through the length of thedegradable block and the chemistry. The ester bonds may also degrade byesterases present in serum, which accelerates degradation. BiodegradablePEG hydrogels can also be fabricated from precursors ofPEG-bis-[2-acryloyloxy propanoate]. As an alternative to linear PEGmacromers, PEG-based dendrimers of poly(glycerol-succinic acid)-PEG,which contain multiple reactive vinyl groups per PEG molecule, can beused. An attractive feature of these materials is the ability to controlthe degree of branching, which consequently affects the overallstructural properties of the hydrogel and its degradation. Degradationwill occur through the ester linkages present in the dendrimer backbone.

The biocompatible, hydrogel-forming polymer can containpolyphosphoesters or polyphosphates where the phosphoester linkage issusceptible to hydrolytic degradation resulting in the release ofphosphate. For example, a phosphoester can be incorporated into thebackbone of a crosslinkable PEG macromer, poly(ethyleneglycol)-di-[ethylphosphatidyl (ethylene glycol) methacrylate](PhosPEG-dMA), to form a biodegradable hydrogel. The addition ofalkaline phosphatase, an ECM component synthesized by bone cells,enhances degradation. The degradation product, phosphoric acid, reactswith calcium ions in the medium to produce insoluble calcium phosphateinducing autocalcification within the hydrogel. Poly(6-aminoethylpropylene phosphate), a polyphosphoester, can be modified withmethacrylates to create multivinyl macromers where the degradation ratewas controlled by the degree of derivitization of the polyphosphoesterpolymer.

Polyphosphazenes are polymers with backbones consisting of nitrogen andphosphorous separated by alternating single and double bonds. Eachphosphorous atom is covalently bonded to two side chains. Thepolyphosphazenes suitable for cross-linking have a majority of sidechain groups which are acidic and capable of forming salt bridges withdi- or trivalent cations. Examples of preferred acidic side groups arecarboxylic acid groups and sulfonic acid groups. Hydrolytically stablepolyphosphazenes are formed of monomers having carboxylic acid sidegroups that are crosslinked by divalent or trivalent cations such asCa²⁺ or Al³⁺. Polymers can be synthesized that degrade by hydrolysis byincorporating monomers having imidazole, amino acid ester, or glycerolside groups. Bioerodible polyphosphazines have at least two differingtypes of side chains, acidic side groups capable of forming salt bridgeswith multivalent cations, and side groups that hydrolyze under in vivoconditions, e.g., imidazole groups, amino acid esters, glycerol andglucosyl. Hydrolysis of the side chain results in erosion of thepolymer. Examples of hydrolyzing side chains are unsubstituted andsubstituted imidizoles and amino acid esters in which the group isbonded to the phosphorous atom through an amino linkage (polyphosphazenepolymers in which both R groups are attached in this manner are known aspolyaminophosphazenes). For polyimidazolephosphazenes, some of the “R”groups on the polyphosphazene backbone are imidazole rings, attached tophosphorous in the backbone through a ring nitrogen atom.

D. Conjugation of Drugs to Hydrogel-Forming Polymer

In some embodiments, one or more anti-inflammatory drugs are covalentlyattached to the hydrogel forming polymer. In these cases, theanti-inflammatory drugs are attached to the hydrogel forming polymer viaa linking moiety that is designed to be cleaved in vivo. The linkingmoiety can be designed to be cleaved hydrolytically, enzymatically, orcombinations thereof, so as to provide for the sustained release of theanti-inflammatory drug in vivo. Both the composition of the linkingmoiety and its point of attachment to the anti-inflammatory agent, areselected so that cleavage of the linking moiety releases either ananti-inflammatory agent, or a suitable prodrug thereof The compositionof the linking moiety can also be selected in view of the desiredrelease rate of the anti-inflammatory agents.

Linking moieties generally include one or more organic functionalgroups. Examples of suitable organic functional groups include secondaryamides (—CONH—), tertiary amides (—CONR—), secondary carbamates(—OCONH—; —NHCOO—), tertiary carbamates (—OCONR—; —NRCOO—), ureas(—NHCONH—; —NRCONH—; —NHCONR—, —NRCONR—), carbinols (—CHOH—, —CROH—),disulfide groups, hydrazones, hydrazides, ethers (—O—), and esters(—COO—, —CH₂O₂C—, CHRO₂C—), wherein R is an alkyl group, an aryl group,or a heterocyclic group. In general, the identity of the one or moreorganic functional groups within the linking moiety can be chosen inview of the desired release rate of the anti-inflammatory agents. Inaddition, the one or more organic functional groups can be chosen tofacilitate the covalent attachment of the anti-inflammatory agents tothe hydrogel forming polymer. In preferred embodiments, the linkingmoiety contains one or more ester linkages which can be cleaved bysimple hydrolysis in vivo to release the anti-inflammatory agents.

In certain embodiments, the linking moiety includes one or more of theorganic functional groups described above in combination with a spacergroup. The spacer group can be composed of any assembly of atoms,including oligomeric and polymeric chains; however, the total number ofatoms in the spacer group is preferably between 3 and 200 atoms, morepreferably between 3 and 150 atoms, more preferably between 3 and 100atoms, most preferably between 3 and 50 atoms. Examples of suitablespacer groups include alkyl groups, heteroalkyl groups, alkylarylgroups, oligo- and polyethylene glycol chains, and oligo- and poly(aminoacid) chains. Variation of the spacer group provides additional controlover the release of the anti-inflammatory agents in vivo. In embodimentswhere the linking moiety includes a spacer group, one or more organicfunctional groups will generally be used to connect the spacer group toboth the anti-inflammatory agent and the hydrogel forming polymer.

In certain embodiments, the one or more anti-inflammatory agents arecovalently attached to the hydrogel forming polymer via a linking moietywhich contains an alkyl group, an ester group, and a hydrazide group. Byway of example, conjugation of the anti-inflammatory agent dexamethasoneto alginate can be via a linking moiety containing an alkyl group, anester group connecting the alkyl group to the anti-inflammatory agent,and a hydrazide group connecting the alkyl group to carboxylic acidgroups located on the alginate. In this embodiment, hydrolysis of theester group in vivo releases dexamethasone at a low dose over anextended period of time.

Reactions and strategies useful for the covalent attachment ofanti-inflammatory agents to hydrogel forming polymers are known in theart. See, for example, March, “Advanced Organic Chemistry,” 5^(th)Edition, 2001, Wiley-Interscience Publication, New York) and Hermanson,“Bioconjugate Techniques,” 1996, Elsevier Academic Press, U.S.A.Appropriate methods for the covalent attachment of a givenanti-inflammatory agent can be selected in view of the linking moietydesired, as well as the structure of the anti-inflammatory agents andhydrogel forming polymers as a whole as it relates to compatibility offunctional groups, protecting group strategies, and the presence oflabile bonds.

E. Anti-Inflammatory and Anti-Proliferative Drugs

Drugs suitable for use in the disclosed compositions are described andcan be identified using disclosed methods. Representative drugs includeglucocorticoids, phenolic antioxidants, anti-proliferative drugs, orcombinations thereof. These are collectively referred to herein as“anti-inflammatory drugs” unless stated otherwise.

Non-limiting examples include steroidal anti-inflammatories.Particularly preferred steroidal anti-inflammatory drugs includedexamethasone, 5-FU, daunomycin, and mitomycin. Anti-angiogenic oranti-proliferative drugs are also useful. Examples include curcuminsincluding monoesters and tetrahydrocurcumin, and drugs such as sirolimus(rapamycin), ciclosporin, tacrolimus, doxorubicin, mycophenolic acid andpaclitaxel and derivatives thereof. In some embodiments, theanti-inflammatory drug is an mTOR inhibitor (e.g., sirolimus andeverolimus). A new antiproliferative drug is biolimus A9, a highlylipophilic, semisynthetic sirolimus analogue with an alkoxy-alkyl groupreplacing hydrogen at position 42-O. Lisofylline is a synthetic smallmolecule with anti-inflammatory properties. In some embodiments, theanti-inflammatory drug is a calcineurin inhibitor (e.g., cyclosporine,pimecrolimus and tacrolimus).

In some embodiments, the anti-inflammatory drug is a synthetic ornatural anti-inflammatory protein. Antibodies specific to select immunecomponents can be added to immunosuppressive therapy. In someembodiments, the anti-inflammatory drug is an anti-T cell antibody(e.g., anti-thymocyte globulin or Anti-lymphocyte globulin), anti-IL-2Rαreceptor antibody (e.g., basiliximab or daclizumab), or anti-CD20antibody (e.g., rituximab).

In preferred embodiments, the one or more anti-inflammatory drugs arereleased from the capsules after administration to a mammalian subjectin an amount effective to inhibit fibrosis of the composition for atleast 30 days, preferably at least 60 days, more preferably at least 90days. In some embodiments, the anti-inflammatory drugs provide spatiallylocalized inhibition of inflammation in the subject without systemicimmunosuppression for at least 10 days, preferably at least 14 days,more preferably at least 30 days. In some embodiments, spatiallylocalized inflammation is detected by measuring cathepsin activity atthe injection sites in the subject. In other embodiments, spatiallylocalized inflammation is detected by measuring reactive oxygen species(ROS) at the injection site in the subject. In some embodiments,systemic immunosuppression is detected by measuring no cathepsinactivity or ROS at control sites in the subject, e.g., sites injectedwith drug-free polymeric particle or hydrogel. Methods for identifying,selecting, and optimizing anti-inflammatory drugs for use in thedisclosed compositions are described below.

The release rate and amounts can be selected in part by modifying drugloading of the polymeric particle. As disclosed herein, higher drugloading can cause a significant initial burst release. This can alsoresult in systemic immunosuppression rather than spatially localizedinhibition of inflammation. In contrast, drug loading levels that aretoo low will not release therapeutically effective amounts ofanti-inflammatory drug.

The optimal drug loading will necessarily depend on many factors,including the choice of drug, polymer, hydrogel, cell, and site ofimplantation. In some embodiments, the one or more anti-inflammatorydrugs are loaded in the polymeric particle at a concentration of about0.01% to about 15%, preferably about 0.1% to about 5%, more preferablyabout 1% to about 3% by weight. In some embodiments, the one or moreanti-inflammatory drugs are encapsulated in the hydrogel at aconcentration of 0.01 to 10.0 mg/ml of hydrogel, preferably 0.1 to 4.0mg/ml of hydrogel, more preferably 0.3 to 2.0 mg/ml of hydrogel.However, optimal drug loading for any given drug, polymer, hydrogel,cell, and site of transplantation can be identified by routine methods,such as those described herein.

F. Biodegradable Polymers for Drug Delivery

The drug-loaded particles containing anti-inflammatory drugs arepreferably formed from a biocompatible, biodegradable polymer suitablefor drug delivery. In general, synthetic polymers are preferred,although natural polymers may be used and have equivalent or even betterproperties, especially some of the natural biopolymers which degrade byhydrolysis, such as some of the polyhydroxyalkanoates.

Representative synthetic polymers include poly(hydroxy acids) such aspoly(lactic acid), poly(glycolic acid), and poly(lactic acid-co-glycolicacid), poly(lactide), poly(glycolide), poly(lactide-co-glycolide),polyanhydrides, polyorthoesters, polyamides, polycarbonates,polyalkylenes such as polyethylene and polypropylene, polyalkyleneglycols such as poly(ethylene glycol), polyalkylene oxides such aspoly(ethylene oxide), polyalkylene terepthalates such as poly(ethyleneterephthalate), polyvinyl alcohols, polyvinyl ethers, polyvinyl esters,polyvinyl halides such as poly(vinyl chloride), polyvinylpyrrolidone,polysiloxanes, poly(vinyl alcohols), poly(vinyl acetate), polystyrene,polyurethanes and co-polymers thereof, derivativized celluloses such asalkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, celluloseesters, nitro celluloses, methyl cellulose, ethyl cellulose,hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutylmethyl cellulose, cellulose acetate, cellulose propionate, celluloseacetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose,cellulose triacetate, and cellulose sulfate sodium salt (jointlyreferred to herein as “synthetic celluloses”), polymers of acrylic acid,methacrylic acid or copolymers or derivatives thereof including esters,poly(methyl methacrylate), poly(ethyl methacrylate),poly(butylmethacrylate), poly(isobutyl methacrylate),poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecylacrylate) (jointly referred to herein as “polyacrylic acids”),poly(butyric acid), poly(valeric acid), andpoly(lactide-co-caprolactone), copolymers and blends thereof As usedherein, “derivatives” include polymers having substitutions, additionsof chemical groups and other modifications routinely made by thoseskilled in the art.

Examples of preferred biodegradable polymers include polymers of hydroxyacids such as lactic acid and glycolic acid, and copolymers with PEG,polyanhydrides, poly(ortho)esters, polyurethanes, poly(butyric acid),poly(valeric acid), poly(lactide-co-caprolactone), blends and copolymersthereof.

Examples of preferred natural polymers include proteins such as albumin,collagen, gelatin and prolamines, for example, zein, and polysaccharidessuch as alginate, cellulose derivatives and polyhydroxyalkanoates, forexample, polyhydroxybutyrate. The in vivo stability of the particles andmicroparticles can be adjusted during the production by using polymerssuch as poly(lactide-co-glycolide) copolymerized with PEG.

In the most preferred embodiment, PLGA is used as the biodegradablepolymer. PLGA particles and microparticles are designed to releasemolecules to be encapsulated or attached over a period of days to weeks.Factors that affect the duration of release include pH of thesurrounding medium (higher rate of release at pH 5 and below due to acidcatalyzed hydrolysis of PLGA) and polymer composition. Aliphaticpolyesters differ in hydrophobicity and that in turn affects thedegradation rate. For example, the hydrophobic poly (lactic acid) (PLA),more hydrophilic poly (glycolic acid) PGA and their copolymers, poly(lactide-co-glycolide) (PLGA) have various release rates. Thedegradation rate of these polymers, and often the corresponding drugrelease rate, can vary from days (PGA) to months (PLA) and is easilymanipulated by varying the ratio of PLA to PGA.

The diameter and porosity of the drug-loaded particle can be optimizedbased on the drug to be delivered and the desired dosage and rate ofrelease. In preferred embodiments, the drug-loaded particle is amicroparticle or a nanoparticle. In more preferred embodiments, thedrug-loaded particle is a particle. The mean diameter of the particlemay be selected and optimized based on the particular drug, dosage, andrelease rate needed. In preferred embodiments, the drug loaded polymericparticles are microparticles having a mean diameter of about 1 μm toabout 100 μm, preferably about 1 μm to about 50 μm, more preferablyabout 1 μm to about 10 μm. In other embodiments, drug loaded polymericparticles are nanoparticles having a mean diameter of about 10 nm toabout 999 nm, including at least about 50 nm, preferably at least about100 nm, more preferably at least about 200 nm. In more preferredembodiments, the drug-loaded polymeric particles have a mean diameterthat is greater than 1 mm, preferably 1.5 mm or greater. In someembodiments, the drug-loaded polymeric particles can be as large at 8 mmin diameter.

G. Capsules

The capsules are two or three layer capsules. Preferably the capsuleshave a mean diameter that is greater than 1 mm, preferably 1.5 mm orgreater. In some embodiments, the capsules can be as large at 8 mm indiameter. For example, the capsule can be in a size range of 1 mm to 8mm, 1 mm to 6 mm, 1 mm to 5 mm, 1 mm to 4 mm, 1 mm to 3 mm, 1 mm to 2mm, 1 mm to 1.5 mm, 1.5 mm to 8 mm, 1.5 mm to 6 mm, 1.5 mm to 5 mm, 1.5mm to 4 mm, 1.5 mm to 3 mm, or 1.5 mm to 2 mm.

The rate of molecules entering the capsule necessary for cell viabilityand the rate of therapeutic products and waste material exiting thecapsule membrane are selected by modulating macrocapsule permeability.Macrocapsule permeability is also modified to limit entry of immunecells, antibodies, and cytokines into the capsule or microcapsule.

It has been shown that, since different cell types have differentmetabolic requirements, the permeability of the membrane has to beoptimized based on the cell type encapsulated in the hydrogel. Thediameter of the capsules or microcapsules is an important factor thatinfluences both the immune response towards the cell capsules as well asthe mass transport across the capsule membrane.

H. Cells

The cell type chosen for encapsulation in the disclosed compositionsdepends on the desired therapeutic effect. The cells may be from thepatient (autologous cells), from another donor of the same species(allogeneic cells), or from another species (xenogeneic). Xenogeneiccells are easily accessible, but the potential for rejection and thedanger of possible transmission of viruses to the patient restrictstheir clinical application. Anti-inflammatory drugs combat the immuneresponse elicited by the presence of such cells. In the case ofautologous cells, the anti-inflammatory drugs reduce the immune responseprovoked by the presence of the foreign hydrogel materials or due to thetrauma of the transplant surgery. Cells can be obtained from biopsy orexcision of the patient or a donor, cell culture, or cadavers.

In some embodiments, the cells secrete a therapeutically effectivesubstance, such as a protein or nucleic acid. In some embodiments, thecells metabolize toxic substances. In some embodiments, the cells formstructural tissues, such as skin, bone, cartilage, blood vessels, ormuscle. In some embodiments, the cells are natural, such as islet cellsthat naturally secrete insulin, or hepatocytes that naturally detoxify.In some embodiments, the cells are genetically engineered to express aheterologous protein or nucleic acid and/or overexpress an endogenousprotein or nucleic acid.

Examples of cells for encapsulation include hepatocytes, islet cells,parathyroid cells, cells of intestinal origin, cells derived from thekidney, and other cells acting primarily to synthesize and secret, or tometabolize materials. A preferred cell type is a pancreatic islet cell.Genetically engineered cells are also suitable for encapsulationaccording to the disclosed methods. In some embodiments, the cells areengineered to secrete blood clotting factors, e.g., for hemophiliatreatment, or to secrete growth hormones for treatment of individualswho are genetically deficient. Alternatively, the cells may beengineered to produce substances that are targeted to cancers or otherdeleterious materials. In some embodiments, the cells are contained innatural or bioengineered tissue. In some embodiments, the cells aresuitable for transplantation into the central nervous system fortreatment of neurodegenerative disease.

The amount and density of cells encapsulated in the disclosedcompositions, such as capsules and microcapsules, will vary depending onthe choice of cell, hydrogel, and site of implantation. In someembodiments, the single cells are present in the hydrogel at aconcentration of 0.1×10⁶ to 4×10⁶ cells/ml, preferably 0.5×10⁶ to 2×10⁶cells/ml. In other embodiments, the cells are present as cellaggregates. For example, islet cell aggregates (or whole islets)preferably contain about 1500-2000 cells for each aggregate of 150 μmdiameter, which is defined as one islet equivalent (IE). Therefore, insome embodiments, islet cells are present at a concentration of100-10000 IE/ml, preferably 200-3,000 IE/ml, more preferably 500-1500IE/ml.

1. Islet Cells

In preferred embodiments, the disclosed compositions contain islet cellsproducing insulin. Methods of isolating pancreatic islet cells are knownin the art. Field et al., Transplantation 61:1554 (1996); Linetsky etal., Diabetes 46:1120 (1997). Fresh pancreatic tissue can be divided bymincing, teasing, comminution and/or collagenase digestion. The isletscan then be isolated from contaminating cells and materials by washing,filtering, centrifuging or picking procedures. Methods and apparatus forisolating and purifying islet cells are described in U.S. Pat. No.5,447,863 to Langley, U.S. Pat. No. 5,322,790 to Scharp et al., U.S.Pat. No. 5,273,904 to Langley, and U.S. Pat. No. 4,868,121 to Scharp etal., The isolated pancreatic cells may optionally be cultured prior toencapsulation or microencapsulation, using any suitable method ofculturing islet cells as is known in the art. See e.g., U.S. Pat. No.5,821,121 to Brothers. Isolated cells may be cultured in a medium underconditions that helps to eliminate antigenic components.

2. Genetically Engineered Cells

In some embodiments, the disclosed compositions contain cellsgenetically engineered to produce a therapeutic protein or nucleic acid.In these embodiments, the cell can be a stem cell (e.g., pluripotent), aprogenitor cell (e.g., multipotent or oligopotent), or a terminallydifferentiated cell (i.e., unipotent). The cell can be engineered tocontain a nucleic acid encoding a therapeutic polynucleotide such miRNAor RNAi or a polynucleotide encoding a protein. The nucleic acid can beintegrated into the cells genomic DNA for stable expression or can be inan expression vector (e.g., plasmid DNA). The therapeutic polynucleotideor protein can be selected based on the disease to be treated and thesite of transplantation. In some embodiments, the therapeuticpolynucleotide or protein is anti-neoplastic. In other embodiments, thetherapeutic polynucleotide or protein is a hormone, growth factor, orenzyme.

III. Methods of Making Capsule

A. Method for Making Bi and Tri-layer Capsules

Triple layer capsules can be made by a tri-fluid coaxial electrosprayingprocess.

Preferably the three layer capsules are formed in an electrosprayingdevice with a nozzle that consists of three separate concentric tubes atits outlet. An exemplary nozzle (100) is illustrated in FIG. 2. A firststream that contains a hydrogel forming material, optionally includingone or more anti-inflammatory drugs or imaging reagents, is pumped intothe innermost tube (110) of the nozzle. A second stream that containshydrogel forming material and islets is pumped into the middle tube(112) of the nozzle. A third stream that contains a hydrogel formingmaterial is delivered to the outermost tube (114).

The three streams meet at the outlet of the nozzle. The relatively highviscosity of the alginate solution prevents any significant intermixingamong the three streams and droplets with three distinct concentriclayers are formed under the electric field. After the droplets fall intothe gel bath which contains divalent ions such as Calcium or Bariumions, crosslinking occurs instantaneously and triple-layer capsules areformed. The triple layer capsules can be made by a tri-fluid coaxialelectrospraying.

For example, a stream that contains anti-inflammatory drugs or imagingreagents is pumped into the innermost tube; a second stream thatcontains islets is pumped into the middle tube; and another stream isdelivered to the outermost tube. The three streams meet at the outlet ofthe nozzle. The relatively high viscosity of the alginate solutionprevents any significant intermixing among the three streams anddroplets with three distinct concentric layers are formed under theelectric field. After the droplets fall into the gel bath which containsdivalent ions such as Calcium or Barium ions, crosslinking occursinstantaneously and triple-layer capsules are formed.

B. Methods for Making Two Layer Capsules

Two-fluid co-axial electro jetting is used for the fabrication ofcore-shell capsules and encapsulation of cells or cell aggregates. Inthe preferred embodiment, the shell fluid consists of a cell-freealginate solution, while the core fluid contains the islets or othertherapeutic cells. The relatively high viscosity of the two fluids andshort interaction time between them prevent their intermixing. Underelectrostatic force, microdroplets with core-shell structures are formedand converted to hydrogel capsules in a gelling bath containing divalentcrosslinking ions.

C. Cell Encapsulation with Polysaccharide Hydrogel

Methods for encapsulating cells in hydrogels are known. In preferredembodiments, the hydrogel is a polysaccharide. For example, methods forencapsulating mammalian cells in an alginate polymer are well known andbriefly described below. See, for example, U.S. Pat. No. 4,352,883 toLim.

Alginate can be ionically cross-linked with divalent cations, in water,at room temperature, to form a hydrogel matrix. An aqueous solutioncontaining the biological materials to be encapsulated is suspended in asolution of a water soluble polymer, the suspension is formed intodroplets which are configured into discrete capsules or microcapsules bycontact with multivalent cations, then the surface of the capsules ormicrocapsules is crosslinked with polyamino acids to form asemipermeable membrane around the encapsulated materials.

The water soluble polymer with charged side groups is crosslinked byreacting the polymer with an aqueous solution containing multivalentions of the opposite charge, either multivalent cations if the polymerhas acidic side groups or multivalent anions if the polymer has basicside groups. The preferred cations for cross-linking of the polymerswith acidic side groups to form a hydrogel are divalent and trivalentcations such as copper, calcium, aluminum, magnesium, strontium, barium,and tin, although di-, tri- or tetra-functional organic cations such asalkylammonium salts, e.g., R₃N+--\∧ ∧/--+NR₃ can also be used. Aqueoussolutions of the salts of these cations are added to the polymers toform soft, highly swollen hydrogels and membranes. The higher theconcentration of cation, or the higher the valence, the greater is thedegree of cross-linking of the polymer. Concentrations from as low as0.005 M have been demonstrated to cross-link the polymer. Higherconcentrations are limited by the solubility of the salt.

The preferred anions for cross-linking of polymers containing basic sidechains to form a hydrogel are divalent and trivalent anions such as lowmolecular weight dicarboxylic acids, for example, terepthalic acid,sulfate ions and carbonate ions. Aqueous solutions of the salts of theseanions are added to the polymers to form soft, highly swollen hydrogelsand membranes, as described with respect to cations.

A variety of polycations can be used to complex and thereby stabilizethe polymer hydrogel into a semi-permeable surface membrane. Examples ofmaterials that can be used include polymers having basic reactive groupssuch as amine or imine groups, having a preferred molecular weightbetween 3,000 and 100,000, such as polyethylenimine and polylysine.These are commercially available. One polycation is poly(L-lysine);examples of synthetic polyamines are: polyethyleneimine,poly(vinylamine), and poly(allyl amine). There are also naturalpolycations such as the polysaccharide, chitosan.

Polyanions that can be used to form a semi-permeable membrane byreaction with basic surface groups on the polymer hydrogel includepolymers and copolymers of acrylic acid, methacrylic acid, and otherderivatives of acrylic acid, polymers with pendant SO₃H groups such assulfonated polystyrene, and polystyrene with carboxylic acid groups.

In a preferred embodiment, alginate capsules are fabricated fromsolution of alginate containing suspended cells using the encapsulator(such as an Inotech encapsulator). In some embodiments, alginates areionically crosslinked with a polyvalent cation, such as Ca²⁺, Ba²⁺, orSr²⁺. In particularly preferred embodiments, the alginate is crosslinkedusing BaCl₂. In some embodiments, the capsules are further purifiedafter formation. In preferred embodiments, the capsules are washed with,for example, HEPES solution, Krebs solution, and/or RPMI-1640 medium.

Cells can be obtained directly from a donor, from cell culture of cellsfrom a donor, or from established cell culture lines. In the preferredembodiments, cells are obtained directly from a donor, washed andimplanted directly in combination with the polymeric material. The cellsare cultured using techniques known to those skilled in the art oftissue culture.

Cell attachment and viability can be assessed using standard techniques,such as histology and fluorescent microscopy. The function of theimplanted cells can be determined using a combination of theabove-techniques and functional assays. For example, in the case ofhepatocytes, in vivo liver function studies can be performed by placinga cannula into the recipient's common bile duct. Bile can then becollected in increments. Bile pigments can be analyzed by high pressureliquid chromatography looking for underivatized tetrapyrroles or by thinlayer chromatography after being converted to azodipyrroles by reactionwith diazotized azodipyrroles ethylanthranilate either with or withouttreatment with P-glucuronidase. Diconjugated and monoconjugatedbilirubin can also be determined by thin layer chromatography afteralkalinemethanolysis of conjugated bile pigments. In general, as thenumber of functioning transplanted hepatocytes increases, the levels ofconjugated bilirubin will increase. Simple liver function tests can alsobe done on blood samples, such as albumin production. Analogous organfunction studies can be conducted using techniques known to thoseskilled in the art, as required to determine the extent of cell functionafter implantation. For example, islet cells of the pancreas may bedelivered in a similar fashion to that specifically used to implanthepatocytes, to achieve glucose regulation by appropriate secretion ofinsulin to cure diabetes. Other endocrine tissues can also be implanted.

The site, or sites, where cells are to be implanted is determined basedon individual need, as is the requisite number of cells. For cellshaving organ function, for example, hepatocytes or islet cells, themixture can be injected into the mesentery, subcutaneous tissue,retroperitoneum, properitoneal space, and intramuscular space.

When desired, the capsules or microcapsules may be treated or incubatedwith a physiologically acceptable salt such as sodium sulfate or likeagents, in order to increase the durability of the capsules ormicrocapsule, while retaining or not unduly damaging the physiologicalresponsiveness of the cells contained in the capsules or microcapsules.By “physiologically acceptable salt” is meant a salt that is not undulydeleterious to the physiological responsiveness of the cellsencapsulated in the capsules or microcapsules. In general, such saltsare salts that have an anion that binds calcium ions sufficiently tostabilize the capsule, without substantially damaging the functionand/or viability of the cells contained therein. Sulfate salts, such assodium sulfate and potassium sulfate, are preferred, and sodium sulfateis most preferred. The incubation step is carried out in an aqueoussolution containing the physiological salt in an amount effective tostabilize the capsules, without substantially damaging the functionand/or viability of the cells contained therein as described above. Ingeneral, the salt is included in an amount of from about 0.1 or 1milliMolar up to about 20 or 100 millimolar, most preferably about 2 to10 millimolar. The duration of the incubation step is not critical, andmay be from about 1 or 10 minutes to about 1 or 2 hours, or more (e.g.,overnight). The temperature at which the incubation step is carried outis likewise not critical, and is typically from about 4° C. up to about37° C., with room temperature (about 21° C.) preferred.

IV. Treatment of Diseases or Disorders

Encapsulated cells can be administered, e.g., injected or transplanted,into a patient in need thereof to treat a disease or disorder. In someembodiments, the disease or disorder is caused by or involves themalfunction of hormone- or protein-secreting cells in a patient. Inthese embodiments, hormone- or protein-secreting cells are encapsulatedand administered to the patient. For example, encapsulated islet cellscan be administered to a patient with diabetes. In other embodiments,the cells are used to repair tissue in a subject. In these embodiments,the cells form structural tissues, such as skin, bone, cartilage,muscle, or blood vessels. In these embodiments, the cells are preferablystem cells or progenitor cells.

1. Diabetes

The potential of using a bioartificial pancreas for treatment ofdiabetes mellitus based on encapsulating islet cells within a semipermeable membrane is extensively being studied by scientists.Microencapsulation protects islet cells from immune rejection and allowsthe use of animal cells or genetically modified insulin-producing cells.

The Edmonton protocol involves implantation of human islets extractedfrom cadaveric donors and has shown improvements towards the treatmentof type 1 diabetics who are prone to hypoglycemic unawareness. However,the two major hurdles faced in this technique are the limitedavailability of donor organs and the need for immunosuppressants toprevent an immune response in the patient's body.

Several studies have been dedicated towards the development ofbioartificial pancreas involving the immobilization of islets cellsinside polymeric capsules. The first attempt towards this aim wasdemonstrated in 1980 by Lim and Sun, Science 210:908 (1980) wherexenograft islet cells were encapsulated inside alginate polylysinemicrocapsules, which resulted in significant in vivo results for severalweeks.

The polymers typically used for islet microencapsulation are alginate,chitosan, polyethylene glycol (PEG), agarose, sodium cellulose sulfateand water insoluble polyacrylates.

2. Cancer

The use of cell encapsulated microcapsules towards the treatment ofseveral forms of cancer has shown great potential. One approachundertaken by researchers is through the implantation of microcapsulescontaining genetically modified cytokine secreting cells. Geneticallymodified IL-2 cytokine secreting non-autologous mouse myoblastsimplanted into mice delay tumor growth with an increased rate ofsurvival of the animals. However, the efficiency of this treatment wasbrief due to an immune response towards the implanted microcapsules.Another approach to cancer suppression is through the use ofangiogenesis inhibitors to prevent the release of growth factors thatlead to the spread of tumors. Genetically modified cytochrome P450expressing cells encapsulated in cellulose sulfate polymers may also beuseful for the treatment of solid tumors.

3. Heart Diseases

While numerous methods have been studied for cell administration toenable cardiac tissue regeneration in patients after ischemic heartdisease, the efficiency of the number of cells retained in the beatingheart after implantation is still very low. A promising approach toovercome this problem is through the use of cell microencapsulationtherapy which has shown to enable a higher cell retention as compared tothe injection of free stem cells into the heart.

Another strategy to improve the impact of cell based encapsulationtechnique towards cardiac regenerative applications is through the useof genetically modified stem cells capable of secreting angiogenicfactors such as vascular endothelial growth factor (VEGF), whichstimulate neovascularization and restore perfusion in the damagedischemic heart.

4. Liver Diseases

Microencapsulated hepatocytes can be used in a bioartificial liverassist device (BLAD). Acute liver failure (ALF) is a medical emergencywhich, despite improvements in modern intensive care, still carries asubstantial mortality rate. In the most severe cases, urgent orthotopicliver transplantation (OLT) currently represents the only chance forsurvival. However, the supply of donor organs is limited and an organmay not become available in time. An effective temporary liver supportsystem would improve the chance of survival in this circumstance bysustaining patients until a donor liver becomes available. Furthermore,the known capacity of the native liver to regenerate following recoveryfrom ALF raises the possibility that the use of temporary liver supportfor a sufficient period of time may even obviate the need for OLT in atleast some cases.

In some embodiments, hepatocytes are encapsulated in capsule ormicrocapsule having an inner core of modified collagen and an outershell of terpolymer of methyl methacrylate (MMA), methacrylate (MAA) andhydroxyethyl methacrylate (HEMA) (Yin C et al., Biomaterials24:1771-1780 (2003)).

Cell lines which have been employed or are currently undergoinginvestigation for use in bioartificial liver support systems includeprimary hepatocytes isolated from human or animal livers, and varioustransformed human cells, such as hepatoma, hepatoblastoma andimmortalized hepatocyte lines.

The present invention will be further understood by reference to thefollowing non-limiting examples.

The examples demonstrate the formation of core-shell alginate basedcapsules and microcapsules, which demonstrate that it is possible toisolate the cells within the hydrogel capsules, preventing the cellsfrom being detected by immune cells in the body (Example 1) and thatlarger, greater than 1 mm in diameter, hydrogel capsules, evoke less ofa fibrotic reaction than the same hydrogel capsules having a smallerdiameter (Example 2).

EXAMPLES Example 1: Preparation of Core-Shell Alginate BasedMicrocapsules

Materials and Methods

Design of Core-Shell Nozzle and Formation of Core-Shell Capsules:

A type of alginate-based hydrogel microcapsules with core-shellstructures using a two-fluid co-axial electro-jetting that is compatiblewith the current alginate-based cell encapsulation protocols wasdeveloped. The composition and thickness of the core and shell can beindependently designed and controlled for many types of biomedicalapplications. FIGS. 3A and 3B are schematic depictions of a conventionalcell encapsulation approach (FIG. 3A) and a two-fluid co-axial electrojetting for core-shell capsules and cell encapsulation (FIG. 3B). FIG.3B shows a schematic of the two-fluid co-axial electro jetting for thefabrication of core-shell capsules and encapsulation of cells or cellaggregates. This two-fluid configuration was used to make core-shellhydrogel capsules and encapsulate living cells.

The shell fluid consists of a cell-free alginate solution, while thecore fluid contains the islets or other therapeutic cells. Therelatively high viscosity of the two fluids and short interaction timebetween them prevent their intermixing. Under electrostatic force,microdroplets with core-shell structures are formed and converted tohydrogel capsules in the gelling bath.

First, to demonstrate the formation of core-shell hydrogel capsules, afluorescently labeled alginate was used to form the shell and anon-labeled alginate was used to form the core. The thicknesses of thecore and shell can be controlled by simply tuning their respective flowrates. For cell encapsulation, the size of the core can then be designedbased on the desired mass of cells per capsule, while that of the shellcan be adjusted according to the requirements of mechanical strength andmass transfer. The compositions of the core and shell are alsoadjustable. This allows independent optimization of the material indirect contact with the encapsulating cells and the one adjacent to thehost immune system when transplanted. For example, an anti-inflammatorydrug or an imaging contrast reagent (e.g., iron oxide nanoparticles) canbe loaded in the shell without contacting the cells in the core. It isalso possible to put a different material such as Matrigel as the coreinside the alginate shell. The core material, which may not be able toform mechanically robust capsules alone, can then provide a preferredlocal environment for the encapsulated cells.

The design of the co-axial nozzle was critical to the stable formationof uniform core-shell structures of the capsules, and completeencapsulation of islets. First, the nozzle must be concentric; anyeccentricity may cause non-uniform flow of the shell fluid surroundingthe core fluid, disturbing the core-shell structure. Second, the nozzlemust be designed in a way that the flow of the shell fluid inside thenozzle is uniform around the core tube. Third, the inner diameter (ID)of the core tube must accommodate the sizes of islets. The co-axialnozzle has a core tube with an ID of 0.014″ and an outer diameter (OD)of 0.022″ and a shell tube with an ID of 0.04″ and an OD of 0.0625″. Thelength of the shell tube that protrudes from the nozzle body is 0.5″ andwas tapered at the outlet tip. The core tube was placed 100 micronsinward relative to the shell tube at the outlet. The flow rates of thecore and shell fluids were independently adjusted by separate syringepumps. The voltage was 6.2 kV and the distance between the nozzle tipand the surface of gelling solution was 1.8 cm.

To optimize encapsulation, the solution concentration of alginate withgiven molecular weight must be optimized. If both the concentrations ofthe core and shell solutions are too small, there will be significantintermixing between the core and shell solutions leading to non-uniformcore—shell structures. For the PRONOVA® SLG20 alginate (Mw approximately75-220,000, FMC BIOPOLYMER®), it was found the optimal concentrationranges were from 0.8% (w/v) to 2% (w/v). All alginate solutions were at1.4% (w/v) and the MATRIGEL® (BD Biosciences) was used at 4 mg/mL in a4° C. cold room to avoid gelling prior to encapsulation. Besides theproperties of the core and shell solutions, optimization of operatingparameters was also important to obtain ideal encapsulation. Typicalflow rates were 0.005 mL/min to 0.1 mL/min for the core flow and 0.1mL/min to 0.5 mL/min for the shell flow.

Rat Islet Isolation and Purification:

Sprague-Dawley rats from Charles River Laboratories weightingapproximately 300 grams were used for harvesting islets. All rats wereanesthetized by a 1:20 Xylazine (10 mg/kg) to Ketamine (150 mg/kg)injection given intraperitoneally, and the total volume of eachinjection was 0.4-0.5 mL depending on the weight of rat. Isolationsurgeries were performed. Briefly, the bile duct was cannulated and thepancreas was distended by an in vivo injection of 0.15% Liberase(Research Grade, Roche) in RPMI 1640 media solution. Rats weresacrificed by cutting the descending aorta and the distended pancreaticorgans were removed and held in 50 mL conical tubes on ice until thecompletion of all surgeries. All tubes were placed in a 37° C. waterbath for a 30 minute digestion, which was stopped by adding 10-15 mL ofcold M199 media with 10% heat-inactivated fetal bovine serum and lightlyshaking. Digested pancreases were washed twice in the sameaforementioned M199 media, filtered through a 450 μm sieve, and thensuspended in a Histopaque 1077 (Sigma)/M199 media gradient andcentrifuged at 1,700 RCF at 4° C. Depending on the thickness of theislet layer that was formed within the gradient, this step was repeatedfor higher purity islets. Finally, the islets were collected from thegradient and further isolated by a series of six gravity sedimentations,in which each supernatant was discarded after four minutes of settling.Purified islets were hand-counted by aliquot under a light microscopeand then washed three times in sterile 1× phosphate-buffered saline.Islets were then washed once in RPMI 1640 media with 10%heat-inactivated fetal bovine serum and 1% penicillin/streptomycin, andcultured in this media overnight for further use.

Liver Tissue Homogenization and Encapsulation:

Liver was obtained from sacrificed rats by dissecting the liver from thehepatic portal, system vessels and connective tissue. It was placed intoa 0.9% NaCl solution and minced using surgical forceps and scissors in apetri dish. These pieces were washed with 0.9% saline twice to removeblood and then dissociated using a gentle MACS Dissociator (MiltenyiBiotec). After dissociation, the tissue sample was filtered through a100 μm cell strainer and subsequently a 40 μm one. This filtration wasrepeated once more to obtain liver tissue cells for encapsulation. Forsingle-fluid encapsulation, 0.06 mL dissociated liver tissue cells weredispersed in 4.5 mL 1.4% SLG20 alginate solution. For the two-fluidcoaxial encapsulation, 0.06 mL dissociated liver cells were dispersed in0.5 mL 1.4% SLG20 alginate solution that was used as the core fluid. Aseparate 4 mL 1.4% alginate solution was used as the shell.

Islets Encapsulation and Transplantation:

Immediately prior to encapsulation, the cultured islets were centrifugedat 1400 rpm for 1 minute and washed with Ca-Free Krebs-Henseleit (KH)Buffer (4.7 mM KCl, 25 mM HEPES, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄×7H₂O, 135mM NaCl, pH approximately 7.4, osmotic pressure approximately 290 mOsm).After the wash, the islets were centrifuged again and all supernatantwas aspirated. The islet pellet was then re-suspended in a 1.4% solutionof SLG20 alginate dissolved in 0.8% NaCl solution at desired isletnumber density. In the case of the regular capsules, 0.27 mL alginatesolution was used for every 1000 islets. For the core-shell capsules,0.03 mL solution for every 1000 islets was used as the core fluid.Another 0.24 mL 1.4% alginate solution without islets was used as theshell fluid. The flow rate for the shell was 0.2 mL/min and that for thecore was 0.025 mL/min. For the same number of islets, the total volumeof the alginate solution used in both regular encapsulation andcore-shell encapsulation was therefore the same. Capsules werecrosslinked using a BaCl₂ gelling solution (20 mM BaCl2, 250 mMD-Mannitol, 25 mM HEPES, pH ˜7.4, osmotic pressure approximately 290mOsm) Immediately after crosslinking, the encapsulated islets werewashed 4 times with HEPES buffer and 2 times with RPMI Medium 1640 andcultured overnight at 37° C. for transplantation. As the islets hadvariable sizes (50-400 μm) and there was an inevitable loss of isletsduring the encapsulation process, the total number of encapsulatedislets were recounted and converted into islet equivalents (IE,normalized to 150 μm size) prior to transplantation.

Immune-competent male C57BL/6 mice were utilized for transplantation. Tocreate insulin-dependent diabetic mice, healthy C57BL/6 mice weretreated with Streptozocin (STZ) by the vendor (Jackson Laboratory, BarHarbor, Me.) prior to shipment to MIT. The blood glucose levels of allthe mice were retested prior to transplantation. Only mice whosenon-fasted blood glucose levels were above 300 mg/dL for two consecutivedays were considered diabetic and underwent transplantation. The micewere anesthetized using 3% isofluorane in oxygen and maintained at thesame rate throughout the procedure. Preoperatively, all mice received a0.05 mg/kg dose of buprenorphine subcutaneously as a pre-surgicalanalgesic, along with 0.3 mL of 0.9% saline subcutaneously to preventdehydration. The abdomens of the mice were shaved and alternatelyscrubbed with betadine and isopropyl alcohol to create a sterile fieldbefore being transferred to the surgical field. A 0.5 mm incision wasmade along the midline of the abdomen and the peritoneum was exposedusing blunt dissection. The peritoneum was then grasped with forceps anda 0.5-1 mm incision was made along the linea alba. A desired volume ofcapsules with predetermined number of islet equivalents were then loadedinto a sterile pipette and transplanted into the peritoneal cavitythrough the incision. The incision was then closed using 5-0 tapertipped polydioxanone (PDS II) absorbable sutures. The skin was thenclosed over the incision using a wound clip and tissue glue.

All animal protocols were approved by the MIT Committee on Animal Careand all surgical procedures and post-operative care were supervised byMIT Division of Comparative Medicine veterinary staff.

Confocal Imaging of Encapsulated Islets:

Pancreatic islets were stained with Hoechst dye (2 μg/mL) andencapsulated in fluorescent, regular capsules or core-shell capsuleswith a fluorescently labeled shell. The capsules were placed onchambered glass coverslips (LabTek) and allowed to settle. Theencapsulated islets were then imaged under a 10× objective using a LaserScanning Confocal Microscope 710 (LSM710). Multiple confocal slices wereimaged from bottom to top of sample to construct a Z-stack. Anorthogonal image and a 3D rendering were performed using LSM browsersoftware to visualize the islet-containing capsules.

Blood Glucose Monitoring:

Blood glucose levels were monitored three times a week following thetransplant surgery. A small drop of blood was collected from the tailvein using a lancet and tested using a commercial glucometer (ClarityOne, Clarity Diagnostic Test Group, Boca Raton, Fla.). Mice withunfasted blood glucose levels below 200 mg/dL were considerednormoglycemic. Monitoring continued until all mice in the experimentalgroup had returned to a hyperglycemic state at which point they wereeuthanized.

Results

Better cell encapsulation using core-shell capsules was demonstratedusing both homogenized rat liver tissue cells and rat pancreatic islets.Microscopic images for both regular capsules and core-shell capsulescontaining islets show a relatively low volume of alginate solution perislet (i.e., 0.27 mL solution for every 1000 islets) used to minimizethe total volume of capsules for transplantation. This is approximatelya third of the volume that is typically used (deVos et al.,Transplantation 1996, 62, 888). For crosslinking, a BaCl₂ solution(deVos et al., Transplantation 1996, 62, 888) was used and the averagecapsule sizes were both around 500 μm. For regular capsules, the isletswere randomly located within the capsules and islets protruding outsidewere frequently observed. In contrast, the islets in the core-shellcapsules were fully encapsulated. The core-shell structure of thecapsules was demonstrated by using a fluorescent alginate in the shelland a non-fluorescent alginate in the core. Fluorescent images where theislets were stained blue (i.e. nuclei staining) showed that in somecases the islets were close to the core-shell interface but stillcompletely encapsulated due to the additional shell layer. It isimportant to note that a confocal microscopy technique was used toconfirm the full encapsulation. Conventional microscopic examination maybe misleading as the relatively dense islet mass may fall at the bottomof the capsule and in line with the objective. In traditionally formedalginate capsules, the islets appeared fully encapsulated underconventional light microscope. The z-series and orthogonal views usingconfocal microscopy revealed however the islets were partially exposedoutside the capsules. In contrast, the core-shell capsules fullyenclosed the islets as viewed from all three directions. Quantitatively,based on examinations of a number of islets-containing capsules usingconfocal microscopy, the regular capsules had about 30% with protrudingislets while the core-shell capsules had none. The hydrogelmicrocapsules with core-shell structures were formed with a shell of analginate partially modified with FITC, while the core is unlabeledalginate or Matrigel. The thicknesses of the shell and the core arecontrolled by tuning their respective flow rates: (a) 0.1 mL/min and 0.1mL/min; (b) 0.15 mL/min and 0.05 mL/min; (c) 0.2 mL/min and 0.02 mL/min.The compositions of the core and shell can also be independentlycontrolled. (d) The shell alginate contains particles of ananti-inflammatory drug, curcumin. The drug particles areself-fluorescent. The shell is fluorescent alginate and the core isnon-fluorescent Matrigel.

Streptozotocin (STZ)—induced diabetic mouse model (Brodsky et al.,Diabetes 1969, 18, 606) was used to evaluate the functionality of thecoaxially encapsulated islets and compare the efficacy with regularcapsules. A comparison was conducted of islets encapsulated in regularcapsules and core-shell capsules. 3D reconstructed confocal fluorescentimages of islets encapsulated in core-shell capsules showed the isletswere stained blue, while the shell was labeled green.

Encapsulated rat islets were transplanted into the peritoneal cavity ofthe diabetic mice and their blood sugar was measured. FIG. 4A shows theblood glucose level of the mice as a function of dayspost-transplantation for both core-shell capsules (diamonds) and regular(circles) capsules. For both types of capsules, a density of 1000 isletsper 0.27 mL alginate solution was used and each mouse was transplantedwith 500 islet equivalents (normalized to 150 μm size). Mice with bloodglucose below 200 mg/dL are considered normoglycemic (Kim et al., LabChip 2011, 11, 246). A couple of days after transplantation, alldiabetic mice became normoglycemic, as expected. However, differencesbetween the two types of capsules started to appear after about 2 weeks,as demonstrated in FIG. 4B. The average blood glucose level for the micereceiving regular capsules went back above 200 mg/dL by 15 days, andthat of the mice receiving core-shell capsules remained below 200 mg/dLup to 50 days. FIG. 4Bb shows the percentage of normoglycemic mice overdays following transplantation. Regular capsules in all mice failed tocontrol the diabetes by 20 days, while the core-shell ones in some ofthe mice did not fail until about 80 days after transplantation,indicating that the core-shell capsules improved the treatmentsignificantly compared with the regular capsules.

In summary, hydrogel microcapsules with core-shell structures and theiruse for improved cell encapsulation and immuno-protection have beenmade. Better islet encapsulation using the core-shell capsules at areduced material volume per islet was demonstrated. Improvedimmune-protection was achieved in a single step by simply confining thecells or cell aggregates in the core region of the capsules. Both thecore-shell structure and better encapsulation were confirmed by confocalmicroscopy. Using a type I diabetic mouse model, it was shown that thecore-shell capsules encapsulating rat islets provided a significantlybetter treatment than the currently used, regular capsules.

Islet microencapsulation represents a promising approach to treat Type Idiabetes and has been a topic of intensive research for decades (Bratlieet al., Adv. Healthcare Mater. 2012, 1, 267). The results from differentresearch groups were often inconsistent. Islet quality and materialproperties such as biocompatibility are certainly critical factors thataffect the outcome of treatment. The quality of encapsulation could alsoinfluence the results and cause inconsistency. The core-shell capsulescan be made in a single step using the same encapsulation protocols thathave been used to make the regular capsules and do no harm to islets. Inaddition to providing better immuno-protection, the opportunity tocontrol the compositions of the shell and the core provide additionalopportunities in microencapsulation. Examples include the replacement ofthe alginate in the core with a different material that may enhancelong-term islet survival. In addition, insulin-producing cells derivedfrom stem cells (Calne et al, Nature Reviews Endocrinology 2010, 6, 173)or adult cells (Zhou, et al., Nature 2008, 455, 627) provide a greatalternative to islets. The core-shell capsules allow not only improvedencapsulation but also greater freedom in the designs of theencapsulating material in the shell and that in direct contact with thecells in the core.

Example 2: Preparation of Large Two-Layer Hydrogel Capsules (diameter>1mm) for Encapsulation of Therapeutic Cells and Cell Aggregates

Materials and Methods

The method of encapsulation includes two general steps: (a) formingdroplets of alginate solution containing islets and (b) conversion ofthe droplets into hydrogel capsules. Mechanical or electrostatic forcescan be used to control the droplet or capsule sizes. The islets can berandomly distributed within the capsules. The islets can also beintroduced in the shell region of the capsules to facilitate the masstransfer. This can be achieved by using a two-fluid encapsulationapproach where one stream of alginate solutions containing islets servesas the shell fluid and a separate stream of cell-free alginate solutionflows in the core. Additional components such as an anti-inflammatorydrug can also be incorporated into the core.

Two types of large (diameter>1 mm) alginate hydrogel capsules for isletsencapsulation were prepared. In the first, islets were dispersedrandomly within the capsules. In the second, islets were purposelyplaced in the shell region of the capsules.

1.5 mm capsules encapsulating rat islets were injected into STZ-induceddiabetic mice. The 500 μm capsules were used as the control and 500 IE'swere used for both sizes of capsules.

Results

The large size (diameter>1 mm) capsules were much less fibrotic andprovided much longer cure than conventional (0-500 μm) capsules. The 500μm capsules were used as the control and 500 IE's were used for bothsizes of capsules. All 5 mice in the 500 μm capsule group failed by 43days (blood glucose above 200 mg/dl for 3 consecutive measurements),while the large capsules lasted much longer. One of the 5 mice failed at43 days, one failed at 75 days, another one failed at 146 days and theremaining two remained cured at 196 days when the experiment wasstopped.

In a separate follow-up study again with 500 IE's, all eight mice in the500 μm capsule group failed by 35 days, while in the 1.5 mm capsulegroup, 1 out of 6 failed at 28 days, 1 failed at 105 days, 1 failed at134 days, another failed at 137 days, and 2 remaining ones stayed curedat 175 days when the experiment was stopped.

See FIG. 5A for 500 micron capsule and FIG. 5B for 1.5 mm capsules.

Analysis of retrieved capsules revealed that the 500 μm capsules wereall fibrosed and clumped but the 1.5 mm capsules were much lessfibrotic.

In summary, large hydrogel capsules, with diameter larger than 1 mm,were made by encapsulating cells or cell aggregates, such as isletsusing materials such as alginate hydrogel. Islets encapsulated inalginate hydrogel capsules can be transplanted to treat Type I diabeteswithout the use of immunosuppressive drugs. One major challenge is thefibrotic reactions to the capsules upon transplantation, whicheventually leads to necrosis of islets and failure of transplant. Thetypical diameter of currently used capsules is relatively small, lessthan 1 mm. It has been discovered that larger capsules have lessfibrosis and could therefore have better disease outcomes for clinicaluses.

Example 3: Comparison of Fibrotic Effects on Large (diameter>1 mm) andSmall (diameter<1 mm) Hydrogel Capsules

The disclosed two or three layer hydrogel capsules are useful for sensoror controlled drug release applications. In some embodiments, thecapsules are designed to resist foreign body responses. Generalcharacteristics of preferred capsules include: a size larger than 1 mmand a spherical shape; a smooth surface with no straight edges; madefrom hydrophilic material; and capsule can be porous, with porespreferably be less than 1 μm in size. Capsules larger than 1 mmeffectively resist macrophage cell adhesion. For example, macrophagecoverage can be less than 30% of total capsule surface area. Thisexample shows the reduced fibrotic effect on capsules larger than 1 mm.Capsules larger than 1 mm effectively resist macrophage and neutrophiladhesion and decrease macrophage and neutrophil recruitment (FIG. 6).For example, macrophage and neutrophil levels were assayed by FACS infibrotic tissue associated with the capsules and was reduced to lessthan 30% of the levels observed with smaller 300 um capsules (FIG. 6).Larger core-shell (multi-layer alginate capsules) were produced usingthe dual nozzle electro-spray method (illustrated in FIG. 1), and theyalso showed similar reduction in macrophage/neutrophil recruitment andadhesion. This holds true for capsules taken from completely healthy aswell as STZ-induced diabetic C57BL/6 mice, throughout many timepointspost-transplantation (e.g., 7, 14, and 28 days, as well as at 1, 6months, and 1 year). Furthermore, fibrosis was measured by visualinspection of cellular adhesion and fibrotic overgrowth upon phasecontrast/brightfield imaging, as well as by confocal imaging and westernblotting for F-actin (cell overgrowth marker) and alpha smooth muscleactin (fibrosis marker). qPCR was also used to determine relative levelsof collagen (1A1) deposition. These examples show the reduced fibroticeffect on capsules larger than 1 mm.

Empty capsules (capsules without encapsulated cells) of different sizeswere produced using SLG100 alginate (PRONOVA). The capsules weretransplanted intraperitoneally in C57BL/6 mice and incubated for 2 weeksbefore retrieval and analysis of host rejection responses (i e, immuneinflammation and fibrosis), as determined by FACS analysis, qPCR,confocal imaging, and/or western blotting. Phase contrast images wereused to assess relative levels of cellular overgrowth and fibrosisacross n=5 individuals per treatment group (i.e., capsule size).Capsules of 300 μm and 500 μm were significantly more fibrotic after 28days than 1100 μm capsules.

Capsules of uniform sizes: small (300 μm), medium (500 μm), and large(1500 μm), were produced using the electrospray technique and incubatedintraperitoneally in C57BL/6 mice and retrieved after 14 days foranalysis. The capsules were examined using several measures of fibrosis(FACS analysis, qPCR, confocal imaging, and/or western blotting).Capsules of 300 μm and 500 μm showed significantly more fibrosis than1500 μm capsules in brightfield and confocal imaging.

The immune response to capsules of different sizes was assessed bymeasuring the percentage of immune cell populations comprised of eithermacrophages or neutrophils (FIG. 6). Capsules of 300 μm, 500 μm, 900 μm,and 1500 μm were incubated for 14 days intraperitoneally in immunecompetent C57BL/6 mice, before samples were retrieved for analysis byFACS. The percentage of macrophages was high for the 300 μm capsules andsteadily declined as the capsule size increased. The percentage ofneutrophils was high for the 300 μm, 500 μm and 900 μm capsules anddropped to nearly the control level for the 1500 μm capsules. Proteinfrom retrieved capsules of 0.3 mm, 0.5 mm, 1 mm, and 1.5 mm capsuleswere also analyzed by western blotting. Western Blotting of smoothmuscle actin (SMA) and β-actin confirmed the same trend of reducedfibrosis with increasing capsule size. The amount of smooth muscle actinand β-actin associated with the different capsule sizes were determinedby Western blot. The signal for smooth muscle actin was normalizedagainst the signal for β-actin. The level of smooth muscle actin was lowfor the 1.5 mm and 1 mm capsules but was much higher for the 1.5 mm and1.0 mm capsules (FIG. 7).

The reduced fibrosis effect remains evident even when the surface areaor volume of the capsules is normalized. Capsules of 300 μm, 500 μm, and800 μm were incubated intraperitoneally in C57BL/6 mice, retrieved after14 days, and imaged using phase contrast microscopy. The amount offibrosis observed was normalized for surface area of the capsules (FIG.8, top) or for volume of the capsules (FIG. 8, bottom). In both cases,the measure of fibrosis was greatest for the 300 μm capsules and lowestfor the 800 μm capsules. Reduced fibrosis for large capsules versussmall capsules was still apparent when large capsules (1000 μm) weretested at 9 times the volume of small capsules (300 μm).

The reduced fibrosis effect of large capsules remains evident across avariety of materials. Capsules of 500 μm and 1500 μm were made of SLG20alginate (PRONOVA®), polystyrene, glass, polycaprolactone (PCL), LF10/60alginate (FMC BIOPOLYMER®), and large 2000 μm Teflon spheres. Thecapsules were incubated in C57BL/6 mice for 14 days, and analyzed byvarious techniques (i.e., qPCR and FACS). Fibrosis was measured usingCD68 (macrophage marker), Ly6g (neutrophil marker), CD11b (myeloid cellmarker), and Collal (fibrosis marker) were assayed by qPCR. With only acouple of exceptions, the markers were expressed at or near the controllevels in all 1500 μm capsules and significantly above the control levelfor all 500 μm capsules (FIG. 9). The exceptions were Ly6g expressionfor polycaprolactone (PCL) capsules and LF10/60 alginate capsules andCD11b expression for LF10/60 alginate capsules. In each of these cases,expression of the marker was higher than the control for the 1500 μmcapsule (but still significantly less than the expression level for the500 μm capsule.

The immune response to capsules of 500 μm and 1500 μm made of thesedifferent materials was also assessed at the cellular level by measuringthe percentage of cell population composed of macrophages andneutrophils by FACS (FIG. 10). The capsules were made of SLG20 alginate(PRONOVA®), polystyrene, glass, polycaprolactone (PCL), and LF10/60alginate (FMC BIOPOLYMER®). The capsules were incubated in C57BL/6 micefor 14 days, and analyzed by various techniques (i.e., qPCR and FACS).In every case the percentage of macrophages and the percentage ofneutrophils was significantly higher for the 500 μm capsules than forthe 1500 μm capsules (FIG. 10).

Modifications and variations of the present invention will be furtherunderstood by reference to the following claims. All references citedherein are specifically incorporated by reference.

We claim:
 1. A population of hydrogel capsules consisting essentially ofhydrogel capsules that have a mean diameter, as measured by confocalmicroscopy, of at least 1.1 mm and less than 8 mm, wherein the hydrogelcapsules: (i) encapsulate mammalian cells selected from the groupconsisting of hepatocytes, islet cells, undifferentiated precursors ofislet cells, partially differentiated precursors of islet cells,parathyroid cells, cells of intestinal origin, insulin-producing cellsderived from stem cells, and insulin-producing cells derived from adultcells; (ii) consist of two hydrogel layers and an optional acellularcore as a third layer, wherein the two hydrogel layers contain a firstinner, hydrogel layer comprising the mammalian cells, and a secondouter, cell-free hydrogel layer, and wherein the first and secondhydrogel layers independently have a three-dimensional open-latticepolymeric structure formed by crosslinking of a biocompatiblehydrogel-forming polymer; and (iii) are spheres or ellipsoids havingsemi-principal axes within 10% of each other.
 2. The population ofhydrogel capsules of claim 1, wherein the mean diameter is at least 1.2mm and less than 8 mm.
 3. The population of hydrogel capsules of claim1, wherein the mean diameter is at least 1.5 mm and less than 8 mm. 4.The population of hydrogel capsules of claim 1, wherein the meandiameter is between 1.5 mm and 6 mm, 1.5 mm and 5 mm, 1.5 mm and 4 mm,1.5 mm and 3 mm, or 1.5 mm and 2 mm.
 5. The population of hydrogelcapsules of claim 1, wherein the biocompatible, hydrogel-forming polymeris selected from the group consisting of polysaccharides, water solublepolyacrylates, polyphosphazenes, poly(acrylic acids), poly(methacrylicacids), copolymers of acrylic acid and methacrylic acid, poly(alkyleneoxides), poly(vinyl acetate), polyvinylpyrrolidones, and blends thereof.6. The population of hydrogel capsules of claim 5, wherein thebiocompatible, hydrogel-forming polymer is selected from the groupconsisting of polysaccharides, polyphosphazenes, poly(alkylene oxides),poly(vinyl acetates), polyvinylpyrrolidones, and blends thereof.
 7. Thepopulation of hydrogel capsules of claim 6, wherein the biocompatible,hydrogel-forming polymer is a polysaccharide selected from the groupconsisting of alginate, chitosan, hyaluronan, and chondroitin sulfate.8. The population of hydrogel capsules of claim 7, wherein thepolysaccharide is an alginate.
 9. The population of hydrogel capsules ofclaim 8, wherein the alginate is cross-linked with barium divalent ions.10. The population of hydrogel capsules of claim 1, wherein the meandiameter is at least 1.2 mm and less than 8 mm and the hydrogel capsulesencapsulating the mammalian cells consist of two hydrogel layers: theinner hydrogel layer comprising the mammalian cells and the secondouter, cell-free hydrogel layer.
 11. The population of hydrogel capsulesof claim 10, wherein the mean diameter is between 1.5 mm and 2 mm. 12.The population of hydrogel capsules of claim 10, wherein thebiocompatible, hydrogel-forming polymer in the second outer, cell-freehydrogel layer comprises an alginate.
 13. The population of hydrogelcapsules of claim 12, wherein the biocompatible, hydrogel-formingpolymer in the first inner, hydrogel layer comprises an alginate. 14.The population of hydrogel capsules of claim 1, wherein thebiocompatible, hydrogel-forming polymer in the second outer, cell-freehydrogel layer comprises an alginate.
 15. The population of hydrogelcapsules of claim 1, wherein the mammalian cells are human cells. 16.The population of hydrogel capsules of claim 1, wherein the mammaliancells are islet cells, or undifferentiated precursors of islet cells, orpartially differentiated precursors of islet cells.
 17. The populationof hydrogel capsules of claim 1, wherein the mammalian cells are isletcells, which comprise beta cells that secrete insulin.
 18. Thepopulation of hydrogel capsules of claim 1, wherein the mammalian cellsare insulin-producing cells derived from stem cells or insulin-producingcells derived from adult cells.
 19. A method of providing functionalcells to an individual in need thereof comprising administering to theindividual an effective amount of a population of hydrogel capsules ofclaim
 16. 20. A method of providing functional cells to an individual inneed thereof comprising administering to the individual an effectiveamount of the population of hydrogel capsules of claim 18.